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Gas-body-based contrast agent enhances vascular bioeffects of 1.09 MHz ultrasound on mouse intestine
Ultrasound in Med. & Biol., Vol. 24, No. 8, pp. 1201–1208, 1998 Copyright © 1998 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/98 $19.00 1 .00
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● Original Contribution GAS-BODY-BASED CONTRAST AGENT ENHANCES VASCULAR BIOEFFECTS OF 1.09 MHz ULTRASOUND ON MOUSE INTESTINE DOUGLAS L. MILLER and RICHARD A. GIES Battelle Pacific Northwest National Laboratory, Richland, WA (Received 5 January 1998; in final form 16 April 1998)
Abstract—Anesthetized hairless mice were exposed to continuous or pulsed 1.09-MHz ultrasound with or without prior injection of a gas-body-based ultrasound contrast agent. Albunext at a dose of 10 mL/kg increased the production of intestinal hyperemia, petechia and hemorrhages by continuous ultrasound. For pulsed ultrasound, with 10 ms pulses and 0.01 duty cycle, petechiae were produced for exposures as low as 1 MPa spatial peak pressure amplitude with added gas bodies. The enhancement of petechiae production was robust for pulsed exposure; for example, at 2.8 MPa, an average of 227 petechiae was obtained with added gas bodies, which was 30 times more than without the agent. The production of petechia was roughly proportional to the dosage of Albunex® for pulsed exposure. Results did not appear to be strongly dependent on pulsing parameters, but long bursts (0.1 s) were somewhat more effective than pulses (10 ms). The observed vascular bioeffects appeared to involve both thermal and nonthermal mechanisms for continuous exposure, but to result primarily from gas-body activation for pulsed exposure. © 1998 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Bioeffects, Petechiae, Hemorrhage, Hyperemia, Cavitation, Mechanical index, Albunex, Contrast agent adverse effects, Gas-body activation.
INTRODUCTION
improved transpulmonary performance, are now under development. In addition to producing useable scattered signals, the strong interaction between the acoustic field and the gas bodies can modify the agents, resulting in an apparent loss of stability of the gas bodies (Vandenberg and Melton 1994), and a diminution of the contrast effect during use (Gottlieb et al. 1995). This destabilization phenomenon appears to allow the contrast agent gas bodies to act as cavitation nuclei, as indicated by the acoustic emissions from ultrasound-exposed agents (Lotsberg et al. 1996; Miller and Bao 1998) and production of the sonochemical hydrogen peroxide (Miller and Thomas 1995). In diagnostic ultrasound, the gas-body activation or cavitation produced by interaction of ultrasound with the contrast agents is exploited by forming images from the scattering and acoustic emissions. The introduction of this controlled form of cavitation into the body for imaging purposes leads to interesting questions with regard to the potential for nonthermal bioeffects. A wide variety of nonthermal biological effects is induced by cavitation in vitro for modest pressure amplitude conditions (Miller et al. 1996). However, cavitation nuclei are normally sparse in the blood of animals, which leads to cavitation thresholds well above pressure amplitudes encountered in diagnostic ultra-
Contrast agents containing stabilized gas bodies have been developed for use in clinical diagnostic ultrasound. The purpose of these agents is to enhance echogenicity of blood-filled regions in a diagnostic image, which allows better characterization of blood vessels, blood flow, and perfusion. The subject of ultrasound contrast agents and their applications has been reviewed recently in some detail (de Jong 1996; Goldberg et al. 1994; Wight 1995). The gas bodies are a few micrometers in diameter, a size that is not only capable of passing through the circulation, but also is able to produce strong scattering of ultrasound in the megaHertz frequency range. For example, Albunex® (Mallinckrodt Medical, St. Louis, MO, USA) consists of numerous small gas bodies that have stable shells of heat-denatured albumin about 15 nm in thickness (Hellebust et al. 1993). The product contains about 2 z 108 mL21 gas bodies between 4 –10 mm in diameter that are stable under refrigeration for several years (Christiansen et al. 1994). Agents with improved characteristics, such as greater persistence and Address correspondence to: D. L. Miller, Mail Stop P7–53, Battelle Pacific Northwest National Laboratory, P. O. Box 999, Richland, WA 99352 USA. E-mail: [email protected] 1201
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sound applications (Williams et al. 1989; Miller and Thomas 1996). Thus, the question arises as to whether or not introduction of a gas-body-based contrast agent into the body can alter this situation, and lead to bioeffects under conditions similar to those observed in vitro. In vitro studies designed to detect ultrasonically induced hemolysis with added contrast agents have been conducted in relatively high cell-concentration suspensions that simulate in vivo conditions. Several of these have indicated a potential for hemolysis at moderate pressure amplitudes in the presence of contrast agents (Brayman et al. 1996; Miller et al. 1997). Newer contrast agents based on perfluorocarbon gases, which improve persistence of the gas bodies, can produce larger effects than observed with air-based agents, particularly for pulsedmode exposures (Miller and Gies 1998b). Brayman et al. (1997) have carefully evaluated the production of hemolysis in 0.4-hematocrit blood supplemented with Albunex® at 1.02, 2.24 and 3.46 MHz and suggest that the hemoglobin released might be sufficient to perturb the hemoglobin-clearing mechanisms of the body under worst-case conditions not too far from clinical practice. Dalecki et al. (1997a) injected Albunex® into mice and detected hemoglobin in the blood after exposure of the heart to ultrasound. Hemolysis amounting to several percent resulted from pulsed 1.15-MHz ultrasound above a threshold of about 3 MPa, but not from 2.35-MHz ultrasound up to 10 MPa. In vivo, cavitation can produce significant vascular damage at high pressure amplitudes, even in the absence of added gas bodies. Extracorporeal shock-wave lithotripsy generates cavitation in vivo, which leads to nonthermal biological effects such as hemorrhage (Coleman and Saunders 1993; Delius 1994). The low shock-wave repetition frequency used in lithotripsy yields low timeaverage power levels and no significant heating. Shockwave–induced hemorrhages have been reported in rat intestine (Chaussy et al. 1982) and in mouse intestine (Raeman et al. 1994). Thresholds for hemorrhage in mouse intestine from lithotripter exposure have been determined to be between 1.6 – 4.0 MPa (i.e., hemorrhages were seen at 4.0 MPa, but not at 1.6 MPa), from experiments using absorbers to vary the pressure amplitude (Miller and Thomas 1995), and to be between 1–3 MPa, from experiments using different positions relative to the shock-wave focus to vary the pressure amplitude (Dalecki et al. 1995a). Intestinal effects ranging from petechiae to gross hemorrhage have also been reported for pulsed and focused ultrasound in the 0.7–3.6-MHz frequency range, with the higher frequencies being much less effective in hemorrhage production (Dalecki et al. 1995b). The addition of contrast agents to the vascular system enhances the vascular damage from lithotripter shock waves, and this phenomenon persists for several
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hours (Dalecki et al. 1997b, 1997c). Other organs also suffer vascular damage. Typically, the mechanism responsible for in vivo biological effects of ultrasound in mammals is simple heating by absorption (Nyborg et al. 1992). Thermal effects vary in different tissues and exposure conditions, and include the induction of petechiae at temperatures as low as 41°C for a few min and hemorrhage of larger vessels at higher temperatures and longer times (Falk 1983). Heating from 1-MHz therapy-mode ultrasound appears to cause petechial hemorrhages in mouse intestine (Miller and Thomas 1994). The ultrasonically induced thermal petechiae involved leakage of blood cells into the lamina propria (a tissue layer within the wall of the intestine), with no evident tissue destruction, and were associated with the occurrence of hyperemia (i.e., engorgement of blood vessels with blood, giving the tissue a reddish coloration). The effects appeared at a spatial peak pressure amplitude of about 0.57 MPa for 1–2 min continuous exposures. Burst modes with 1-ms bursts repeated at 2– 4-ms intervals produced similar effects, which were comparable for comparable spatial average intensities. The petechiae observed to arise from heating with therapy-mode ultrasound were quite different from the intestinal hemorrhages induced by lithotripter shock waves. The shock-wave–induced hemorrhages involved blood flowing into the lumen of the intestine, with histologically obvious tissue destruction and clotting (Miller and Thomas 1995). The observation that ultrasound could produce vascular damage to the intestine both by heating (Miller and Thomas 1994) and by cavitation (Miller and Thomas 1995) led to an examination of the possibility of an interaction between the two mechanisms (Miller and Gies 1998a). Focused ultrasound at 400 kHz that was continuous or pulsed with 100 ms pulses, was applied to mice in a temperature-controlled water bath. Production of petechiae, which appeared to be primarily due to heating, and hemorrhages, which appeared to be primarily due to cavitation, were examined for various exposure conditions designed to elicit separate or combined operation of the two mechanisms. Petechia (up to about 100) occurred above 0.28 MPa (2.6 W cm22) for 1000 s continuous exposure at 37°C, and the threshold increased to 6.5 MPa (1.4 W cm temporal average) for 1000-s pulsed exposure (0.001 duty factor). Hemorrhages (up to about 10) were seen above 0.65 MPa for continuous exposure, and above 1.6 MPa for 1000-s pulsed exposure (0.001 duty factor). If a brief continuous exposure was broken into a low duty-factor pulsed exposure, the petechiae production decreased, but the number of hemorrhages increased. More petechia were induced at 42°C bath temperature relative to 32°C or 37°C, and the hemorrhage effect was somewhat enhanced by elevated tem-
Contrast agent enhances bioeffects ● D. L. MILLER and R. A. GIES
perature. Although some interaction could be discerned, for the most part, the two mechanisms appeared to act independently to produce vascular bioeffects on mouse intestine. The mouse intestine has become a valuable model system for ultrasound bioeffects research, and has been characterized in regard to vascular damage from both heating and cavitation under normal conditions. The intestines are especially convenient for studies of vascular damage because petechiae and hemorrhages can be counted by simple visual inspection without recourse to histology. The purpose of this present study was to utilize this model system to assess the possible association of vascular damage with ultrasonic activation of ultrasound contrast agent gas bodies circulating in the blood. With gas bodies present in the blood, the potential arises for damage, such as petechiae, to occur as a consequence of gas-body activation in addition to the thermal petechiae and nonthermal hemorrhage seen without added gas bodies. The addition of Albunex® to the circulation led to a complex interplay of thermal and nonthermal effects for different exposure conditions. METHODS Handling and treatment of the hairless male mice (Charles River, SK-H1) have been described previously (Miller and Thomas 1994), and all animal procedures were in accordance with institutional Animal Care Committee guidelines. A mouse was anesthetized with an intramuscular (IM) injection of 100 mg/kg ketamine (Ketaset®, Aveco Co., Fort Dodge, IA, USA) and 20 mg/kg xylazine (Rompun®, Mobay Corp., Shawnee, KA, USA). The animal was then mounted on a plastic board over a 2.5 cm diameter hole to allow passage of the beam, and placed at a 45° angle into a 37°C water bath. A rectal temperature probe (RET-3, Harvard Apparatus) was used to assure the equilibration of the mouse temperature with the bath temperature. Either Albunex® ultrasound contrast agent, or a gas-body–free blank, was introduced into the mouse by retro-orbital injection with a 25-G needle at a dosage of 10 mL/kg for most experiments immediately prior to exposure. Retro-orbital injection was chosen for introducing the agents into the blood, due to the rapid and positive injection afforded by this method (Bao et al. 1990). For this study, the Albunex® was purchased from Mallinckrodt Medical, St. Louis, MO, USA. The blank was a sterile 5% solution of human serum albumin (Sigma) in isotonic saline. When different dosages were used, a fraction of the blank agent was replaced with Albunex®, so that the injected volume was always the same. For exposure, an air-backed 2.5 cm diameter transducer was aimed upward at the beam hole at a 45° angle
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and positioned at 5.5 cm from the ventral surface of the mouse. The transducer was driven at 1.09 MHz, which was approximately its fundamental thickness resonance, by a signal generator (Model 3314A, Hewlett-Packard Co., Santa Clara, CA, USA) and amplifier (Model A-500, Electronic Navigation Industries, Rochester, NY, USA). A second oscillator (Model 33120A, Hewlett Packard) was used to gate the signal generator for some experiments. The exposure bath was filled with purified water, which was degassed for 1 h before each exposure session and filtered continuously through a 0.2 mm filter to minimize cavitation in the bath water. The ultrasound field was calibrated by measurements with a bilaminar shielded hydrophone with 0.5 mm sensitive spot (Marconi type Y-34-3598, National Physical Laboratory, Middlesex, UK). The 26 dB beam width was 13.4 mm at the mouse. Exposures were varied in 3-dB increments by linear extrapolation from the calibration at about 0.4 MPa. The influence of finite amplitude distortion on the beam was checked and the peak positive and peak negative differed by only about 20%, even for the highest exposures used in this study. The mean amplitude is specified to characterize the exposure level. After exposure, the mouse was removed from the bath and holder, and euthanized by CO2 asphyxiation at 5 min postexposure. The mouse was then dissected and the intestines were evaluated, as described previously (Miller and Gies 1998a). The number of petechiae, which were visible as small bright-red spots, extent of hyperemia, and the number of hemorrhages, which were visible as dark red blood in segments of the intestinal lumen, were evaluated and recorded. The petechiae, when present, had the same visual appearance regardless of the exposure mode, and no distinction could be made during scoring as to whether these lesions were caused by heating or by gas-body activation. Results are reported as the mean and standard error of 5 repeated measurements in different mice. Student’s t-tests were performed to compare results, with statistically significant difference assumed at p , 0.05 (two-sided test). Thresholds were estimated as the mean of the lowest pressure amplitude step with a statistically significant increase in a measure relative to 9 sham exposures, and the next lower step in the dose–response progression. Temperatures were measured in 4 separate mice (different from those used to study the bioeffects) using a 0.3-mm diameter thermocouple probe (model HYP-1, Omega Engineering) inserted into the abdomen immediately beneath the abdominal wall at the approximate position of the spatial peak intensity in the beam. These measurements should be considered estimates of the true maximum temperature elevations because the probe was not scanned to locate the exact maximum. Experiments were planned with continuous or pulsed-mode exposure
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with the goal of separating the effects of heating and gas-body activation. RESULTS Results for continuous 100-s exposures are shown in Fig. 1A, B, C for a range of pressure amplitudes. Hyperemia, an effect of heating, appears at about 0.5 MPa, and rises dramatically at 0.7 MPa, which was the maximum continuous-wave exposure level employed. Exposures with added contrast agent gas bodies enhanced this effect, for example, by a factor of 4 at 0.7 MPa. Petechiae were produced in the mouse intestine with the blank agent, and this effect was also enhanced by addition of gas bodies, for example, by a factor of about 5 at 0.7 MPa. No significant hemorrhage production occurred in the intestines for mice injected with the blank agent, but this effect was induced with Albunex® treatment. The thresholds determined for this exposureresponse experiment are listed in Table 1. For continuous exposure at 0.5 MPa, temperature elevations in 4 mice averaged 9.8°C (2.8°C SD). The thermal effects generally require some time to develop, but cavitational effects can presumably occur very quickly. The influence of the exposure duration on petechiae generation is shown in Fig. 2 for exposures with or without gas bodies. Hyperemia and hemorrhages were not appreciable except at 100 s (shown in Fig. 1). With Albunex®, significant petechia were produced at 10 s and 100 s. At 100 s, the petechiae production without added gas bodies was significantly different both from sham treatment and from the exposure with added gas bodies. Results for 100-s pulsed-mode exposures are shown in Fig. 1 for a range of pressure amplitudes of 10 ms pulses with a 1-ms pulse-repetition period (PRP). The pulse-mode exposure reduces the heating, and essentially no hyperemia was found for these exposures up to 2.8 MPa. Petechiae production was also minimal without added gas bodies, but hemorrhages began to appear at the highest exposure. Exposures with the contrast agent gas bodies began to yield significant numbers of petechiae at 1 MPa, but hemorrhage was seen only at the highest exposure. The petechiae seen with added gas bodies with pulsed exposure had the same visual appearance as the petechiae seen after the continuous exposures. The enhancement in the production of petechiae with added gas bodies was robust; for example, at 2.8 MPa, an average of 227 petechiae was produced with added gas bodies, a 30-fold increase. The thresholds determined for this exposure-response experiment are listed in Table 1. At 1.0 MPa with Albunex®, estimates of the temperature elevation from 4 mice averaged 0.6°C (0.3°C SD). The pronounced enhancement of petechiae produc-
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tion for pulse-mode exposure depends on the dosage of Albunex®, as shown in Fig. 3 for 2-MPa pulses. The line was fitted to the plotted data points by linear regression, with a slope of 10.4 petechia per mL/kg and an intercept of 6.4 petechia (r 2 5 0.97). Although there is a clear trend for increasing numbers of petechiae, statistically significant increases above the effect obtained with the blank agent were obtained only for the 5 mL/kg and 10 mL/kg doses. However, the petechia effect was significant relative to sham exposure for 0.1, 0.2, 1.0, 5.0 and 10 mL/kg contrast agent doses (but not for 0, 0.5 or 2.0 mL/kg). Hemorrhage and hyperemia were not appreciable in this series of exposures. The pulsing variables of pulse duration and duty factor were explored to provide a more complete picture of the role of pulsing in the effects with and without added contrast agent gas bodies. Results for 10-ms pulses (1 ms PRP) are compared with 1 ms pulses (100 ms PRP) in Fig. 4. Essentially, no effect was seen without Albunex® (10 mL/kg), and no difference was found in the petechiae counts for the two pulse durations. The range of pulse durations was extended, all with a 0.01 duty factor, further, to explore this variable. The results are shown in Fig. 5 for 10-ms to 100-ms pulses. All the gas-body agent results were significantly different from the blank agent results, except for the 10-ms pulse duration. The 100-ms duration pulses, which had a 10-s PRP giving only 10 pulses during the 100-s exposure, produced significantly more petechiae than the 1-ms or shorter pulses. The duty factor was also important because more closely spaced pulses increase the temporal average intensity (related to heating), as shown in Fig. 6 for 2-MPa, 10-ms pulses. The gas-body agent produced significantly more petechiae than the blank agent, but this enhancement declined greatly for the 0.001 duty cycle (10-ms PRP, 1000-s duration). The petechiae effect was also significantly different from sham exposure for the blank agent, at 0.1 duty factor. DISCUSSION The results of this study revealed a complex interplay of heating and gas-body activation in the etiology of ultrasound-induced intestinal effects. For continuous exposure, petechiae and hyperemia were induced even for the blank agent, and these effects were enhanced when gas bodies were injected (see Fig. 1). Because hyperemia was associated with the effects, heating must be a factor in production of the effects, as was noted previously for similar exposure conditions (Miller and Thomas 1994). The rapid decline in the effect for shorter exposure durations (see Fig. 2) also may be indicative of an important thermal role in the enhancement. The enhancement of the effects by the addition of contrast-agent gas
Contrast agent enhances bioeffects ● D. L. MILLER and R. A. GIES
Fig. 1. Results for average numbers (standard error bars) of (a) hyperemic length, (b) Petechiae and (c) Hemorrhages induced in intestines of mice exposed to continuous (circles) or pulsed (diamonds) ultrasound. Prior to exposure, mice received an injection of gas-body contrast agent (solid symbols) or blank agent without gas bodies (open symbols).
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Table 1. Thresholds, as defined in Methods, for petechiae and hemorrhages for each of the exposure conditions involving an exposure-response series without or with added gas bodies (1 GB). Threshold (MPa) Exposure condition Continuous Continuous 1 GB Pulsed 10 ms Pulsed 10 ms 1 GB Pulsed 1 ms Pulsed 1 ms 1 GB
Threshold (W cm22)
Petechia Hemorrhage Petechia Hemorrhage 0.4 0.4 1.2 0.8 1.7 0.4
ND 0.4 2.4 2.4 ND 1.7
5.3 5.3 0.48 0.21 0.96 0.05
ND 5.3 1.9 1.9 ND 0.96
The thresholds are specified in terms of the spatial peak pressure amplitude and the spatial peak; temporal average intensity calculated using the 0.01 duty factor for the pulsed modes. ND: not determinable from the data.
bodies may reflect an increase in absorption and heating, or the mechanical action of the gas bodies (or both). Theoretical considerations indicate that the presence of gas bodies or bubbles in tissue can increase the absorption and heating resulting from ultrasound exposure (Coakley and Nyborg 1978; Wu 1998). The significant production of hemorrhage into the intestinal lumen with added contrast agent suggests that the gas bodies may be initiating vigorous cavitation activity, because this effect was previously associated with lithotripter shock-wave exposure (Miller and Thomas 1995). For pulse-mode exposure, heating was minimized, hyperemia was avoided, and the petechia appeared with the addition of gas bodies at levels for which little or no effect was detectable for the blank agent. The cause of
Fig. 2. Results for average numbers (standard error bars) of petechiae for 0.7-MPa continuous exposures of varying duration with (solid circles) or without (open circles) added gas bodies.
Fig. 3. The average numbers (standard error bars) of petechiae obtained for various doses of contrast agent after exposure to 2-MPa pulsed ultrasound. The line was fitted to the data by linear regression (see text).
this effect was presumably the mechanical rupture of capillaries by the ultrasonically-activated gas bodies. The petechiae had the same visual appearance for continuous or pulsed exposures, and it was not possible to categorize these lesions as thermal or nonthermal in origin by simple visual inspection. The threshold for this pulsemode effect, 0.8 MPa peak pressure amplitude or 210 mW/cm2 SPTA intensity for the 10-ms pulses, lies within the range of parameters associated with diagnostic ultrasound.
Fig. 4. Results for average numbers (standard error bars) of petechiae produced by pulsed mode exposure for 10 ms or 1-ms pulses with 0.01 duty factor with or without added gas bodies.
Contrast agent enhances bioeffects ● D. L. MILLER and R. A. GIES
Fig. 5. Petechiae induced by 2-MPa pulsed ultrasound with various pulse durations, 0.01 duty cycle and 100-s duration, with and without added gas bodies.
In this study, the contrast agent was delivered as a per-kg dosage by retro-orbital injection. Unfortunately, the actual dosage of intact gas bodies reaching the general circulation and persisting until exposure (about 1 min) is unknown. This problem pertains to all small animal experimentation; for example, similar problems were encountered for tail-vein injection (Dalecki et al. 1997a). As a rough perspective, the dosage used for most experiments in this study was 10 mL/kg, which would extrapolate to a 1000-mL injection into a 100-kg human. The petechiae effect decreased roughly in proportion to the dosage (see Fig. 3). More realistic dosages, such as 0.1 mL/kg (10 mL in the 100-kg human) produced sta-
Fig. 6. Petechiae induced by 2-MPa pulsed ultrasound with 10 ms pulses and three different duty factors, with or without added contrast agent. The exposure duration was also varied to maintain a constant total on-time of 1 s.
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tistically significant, but greatly reduced, numbers of petechiae in this study. The results of pulsed-mode exposure in this study are different from those of the earlier study (Miller and Thomas 1994), which involved closely spaced 1-ms pulses. This difference was presumably due to the closely spaced pulses, which produced significant heating in the previous study, relative to the widely spaced pulses, which produced much less heating in this study. Pulsing parameters did not appear to be a strong factor in the exposure outcome; for example, results for 10-ms and 1-ms pulses were very similar (see Fig. 4). However, the threshold with gas bodies was lower for the longer pulse (50 mW/cm2, see Table 1). Very long, widely spaced pulses were more effective than short pulses at the same duty cycle (see Fig. 5). This phenomenon is puzzling because the longest pulses had a PRP of 10 s. This time was sufficient for the small gas bodies to dissolve and vanish after destabilization by the exposure and, thus, a diminution of the effect might have been anticipated for this condition. One explanation for this observation is that the long interpulse duration may have allowed inflow of a fresh supply of gas bodies from unexposed regions of the body. This concept has also been applied in diagnostic procedures, for which the ultrasound beam can reduce contrast (Porter et al. 1996). If unusually long times are allowed between scans, then the contrast effect returns. In conclusion, the addition of contrast agent during in vivo ultrasound exposure enhanced the production of vascular damage in intestinal tissue. In particular, added gas bodies yielded petechiae production under exposure conditions for which heating was insufficient to produced this effect. Thus, the petechia effect, as observed by visual inspection, can be produced by ultrasonic activation of blood-borne gas bodies, as well as by heating. The intestine was utilized for this study, because it is a previously examined model system, but other tissues would presumably also be effected. For example, petechiae were noted in the abdominal skin and muscle layers for many exposures yielding intestinal petechiae. The petechiae and hemorrhage effects were increased with added gas bodies for continuous exposure, which involved significant heating and hyperemia. For pulsed exposure, petechiae were produced for conditions yielding essentially no effect without the agent, presumably by the nonthermal mechanical action of the gas bodies. In this study, effects associated with the contrast agent were emphasized by utilizing relatively high doses of the agent, and effects decreased for lower dosages. Because the addition of gas bodies to the circulation introduces an extrinsic potential for activation of nonthermal mechanisms for bioeffects, some increased effectiveness might
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be expected for any dosage when the gas bodies interact with ultrasound. Acknowledgements—This investigation was supported by PHS Grant CA42947 awarded by the National Institutes of Health, DHHS.
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Volume 24, Number 8, 1998 Gottlieb S, Ernst A, Meltzer R. Effect of pressure on echocardiographic videodensity from sonicated albumin: an in vitro model. J Ultrasound Med 1995;14:109 –116. Hellebust H, Christiansen C, Skotland T. Biochemical characterization of air-filled albumin microspheres. Biotechnol Appl Biochem 1993; 18:227–237. Lotsberg O, Hovem JM, Aksum B. Experimental observation of subharmonic oscillations in Infoson bubbles. J Acoust Soc Am 1996; 99:1366 –1369. Miller DL, Bao S. The relationship of scattered subharmonic, 3.3 MHz fundamental and second harmonic signals to damage of monolayer cells by ultrasonically activated Albunex. J Acoust Soc Am 1998; 103:1183–1189. Miller DL, Gies RA. The interaction of ultrasonic heating and cavitation in vascular bioeffects on mouse intestine. Ultrasound Med Biol 1998a:24:123–128. Miller DL, Gies RA. Enhancement of ultrasonically induced hemolysis by perfluorocarbon-based compared to air-based echo-contrast agents. Ultrasound Med Biol 1998b;24:285–292. Miller DL, Thomas RM. Heating as a mechanism for ultrasonically induced petechial hemorrhages in mouse intestine. Ultrasound Med Biol 1994;20:493–503. Miller DL, Thomas RM. Thresholds for hemorrhages in mouse skin and intestine produced by lithotripter shockwaves. Ultrasound Med Biol 1995;21:294 –257. Miller DL, Thomas RM. Contrast-agent gas bodies enhance hemolysis induced by lithotripter shock waves and high-intensity focused ultrasound in whole blood. Ultrasound Med Biol 1996;22:1089 – 1095. Miller DL, Gies RA, Chrisler WB. Ultrasonically induced hemolysis at high cell and gas body concentrations in a thin-disc exposure chamber. Ultrasound Med Biol 1997;23:625– 633. Miller MW, Miller DL, Brayman AA. A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 1996;22:1131–1154. Nyborg WL, Carson PL, Miller MW, Dunn F, Ziskin MC, Miller DL, Thompson HE, Edwards M. Exposure criteria for medical diagnostic ultrasound: I. Criteria based on thermal mechanisms. NCRP report No. 113. Bethesda, MD: National Council on Radiation Protection and Measurements, 1992. Porter TR, Xie F, Li S, D’Sa A, Rafter P. Increased ultrasound contrast and decreased microbubble destruction rates with triggered ultrasound imaging. J Am Soc Echocardiogr 1996;9:599 – 605. Raeman CH, Child SZ, Dalecki D, Mayer R, Parker KJ, Carstensen EL. Damage to murine kidney and intestine from exposure to the fields of a piezoelectric lithotripter. Ultrasound Med Biol 1994;20:589 – 594. Vandenberg BF, Melton HE. Acoustic lability of albumin microspheres. J Am Soc Echocardiog 1994;7:582–589. Wight PS. Microbubble contrast agents. Adv Admin Radiol 1995;5: 2–5. Williams AR, Delius M, Miller DL, Schwarze W. Investigation of cavitation in flowing media by lithotripter shock waves both in vitro and in vivo. Ultrasound Med Biol 1989;15:53– 60. Wu J. Temperature rise generated by ultrasound in the presence of contrast agent. Ultrasound Med Biol 1998;24:267–274.
PII S031-5629(98)00063-5
● Original Contribution GAS-BODY-BASED CONTRAST AGENT ENHANCES VASCULAR BIOEFFECTS OF 1.09 MHz ULTRASOUND ON MOUSE INTESTINE DOUGLAS L. MILLER and RICHARD A. GIES Battelle Pacific Northwest National Laboratory, Richland, WA (Received 5 January 1998; in final form 16 April 1998)
Abstract—Anesthetized hairless mice were exposed to continuous or pulsed 1.09-MHz ultrasound with or without prior injection of a gas-body-based ultrasound contrast agent. Albunext at a dose of 10 mL/kg increased the production of intestinal hyperemia, petechia and hemorrhages by continuous ultrasound. For pulsed ultrasound, with 10 ms pulses and 0.01 duty cycle, petechiae were produced for exposures as low as 1 MPa spatial peak pressure amplitude with added gas bodies. The enhancement of petechiae production was robust for pulsed exposure; for example, at 2.8 MPa, an average of 227 petechiae was obtained with added gas bodies, which was 30 times more than without the agent. The production of petechia was roughly proportional to the dosage of Albunex® for pulsed exposure. Results did not appear to be strongly dependent on pulsing parameters, but long bursts (0.1 s) were somewhat more effective than pulses (10 ms). The observed vascular bioeffects appeared to involve both thermal and nonthermal mechanisms for continuous exposure, but to result primarily from gas-body activation for pulsed exposure. © 1998 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Bioeffects, Petechiae, Hemorrhage, Hyperemia, Cavitation, Mechanical index, Albunex, Contrast agent adverse effects, Gas-body activation.
INTRODUCTION
improved transpulmonary performance, are now under development. In addition to producing useable scattered signals, the strong interaction between the acoustic field and the gas bodies can modify the agents, resulting in an apparent loss of stability of the gas bodies (Vandenberg and Melton 1994), and a diminution of the contrast effect during use (Gottlieb et al. 1995). This destabilization phenomenon appears to allow the contrast agent gas bodies to act as cavitation nuclei, as indicated by the acoustic emissions from ultrasound-exposed agents (Lotsberg et al. 1996; Miller and Bao 1998) and production of the sonochemical hydrogen peroxide (Miller and Thomas 1995). In diagnostic ultrasound, the gas-body activation or cavitation produced by interaction of ultrasound with the contrast agents is exploited by forming images from the scattering and acoustic emissions. The introduction of this controlled form of cavitation into the body for imaging purposes leads to interesting questions with regard to the potential for nonthermal bioeffects. A wide variety of nonthermal biological effects is induced by cavitation in vitro for modest pressure amplitude conditions (Miller et al. 1996). However, cavitation nuclei are normally sparse in the blood of animals, which leads to cavitation thresholds well above pressure amplitudes encountered in diagnostic ultra-
Contrast agents containing stabilized gas bodies have been developed for use in clinical diagnostic ultrasound. The purpose of these agents is to enhance echogenicity of blood-filled regions in a diagnostic image, which allows better characterization of blood vessels, blood flow, and perfusion. The subject of ultrasound contrast agents and their applications has been reviewed recently in some detail (de Jong 1996; Goldberg et al. 1994; Wight 1995). The gas bodies are a few micrometers in diameter, a size that is not only capable of passing through the circulation, but also is able to produce strong scattering of ultrasound in the megaHertz frequency range. For example, Albunex® (Mallinckrodt Medical, St. Louis, MO, USA) consists of numerous small gas bodies that have stable shells of heat-denatured albumin about 15 nm in thickness (Hellebust et al. 1993). The product contains about 2 z 108 mL21 gas bodies between 4 –10 mm in diameter that are stable under refrigeration for several years (Christiansen et al. 1994). Agents with improved characteristics, such as greater persistence and Address correspondence to: D. L. Miller, Mail Stop P7–53, Battelle Pacific Northwest National Laboratory, P. O. Box 999, Richland, WA 99352 USA. E-mail: [email protected] 1201
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sound applications (Williams et al. 1989; Miller and Thomas 1996). Thus, the question arises as to whether or not introduction of a gas-body-based contrast agent into the body can alter this situation, and lead to bioeffects under conditions similar to those observed in vitro. In vitro studies designed to detect ultrasonically induced hemolysis with added contrast agents have been conducted in relatively high cell-concentration suspensions that simulate in vivo conditions. Several of these have indicated a potential for hemolysis at moderate pressure amplitudes in the presence of contrast agents (Brayman et al. 1996; Miller et al. 1997). Newer contrast agents based on perfluorocarbon gases, which improve persistence of the gas bodies, can produce larger effects than observed with air-based agents, particularly for pulsedmode exposures (Miller and Gies 1998b). Brayman et al. (1997) have carefully evaluated the production of hemolysis in 0.4-hematocrit blood supplemented with Albunex® at 1.02, 2.24 and 3.46 MHz and suggest that the hemoglobin released might be sufficient to perturb the hemoglobin-clearing mechanisms of the body under worst-case conditions not too far from clinical practice. Dalecki et al. (1997a) injected Albunex® into mice and detected hemoglobin in the blood after exposure of the heart to ultrasound. Hemolysis amounting to several percent resulted from pulsed 1.15-MHz ultrasound above a threshold of about 3 MPa, but not from 2.35-MHz ultrasound up to 10 MPa. In vivo, cavitation can produce significant vascular damage at high pressure amplitudes, even in the absence of added gas bodies. Extracorporeal shock-wave lithotripsy generates cavitation in vivo, which leads to nonthermal biological effects such as hemorrhage (Coleman and Saunders 1993; Delius 1994). The low shock-wave repetition frequency used in lithotripsy yields low timeaverage power levels and no significant heating. Shockwave–induced hemorrhages have been reported in rat intestine (Chaussy et al. 1982) and in mouse intestine (Raeman et al. 1994). Thresholds for hemorrhage in mouse intestine from lithotripter exposure have been determined to be between 1.6 – 4.0 MPa (i.e., hemorrhages were seen at 4.0 MPa, but not at 1.6 MPa), from experiments using absorbers to vary the pressure amplitude (Miller and Thomas 1995), and to be between 1–3 MPa, from experiments using different positions relative to the shock-wave focus to vary the pressure amplitude (Dalecki et al. 1995a). Intestinal effects ranging from petechiae to gross hemorrhage have also been reported for pulsed and focused ultrasound in the 0.7–3.6-MHz frequency range, with the higher frequencies being much less effective in hemorrhage production (Dalecki et al. 1995b). The addition of contrast agents to the vascular system enhances the vascular damage from lithotripter shock waves, and this phenomenon persists for several
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hours (Dalecki et al. 1997b, 1997c). Other organs also suffer vascular damage. Typically, the mechanism responsible for in vivo biological effects of ultrasound in mammals is simple heating by absorption (Nyborg et al. 1992). Thermal effects vary in different tissues and exposure conditions, and include the induction of petechiae at temperatures as low as 41°C for a few min and hemorrhage of larger vessels at higher temperatures and longer times (Falk 1983). Heating from 1-MHz therapy-mode ultrasound appears to cause petechial hemorrhages in mouse intestine (Miller and Thomas 1994). The ultrasonically induced thermal petechiae involved leakage of blood cells into the lamina propria (a tissue layer within the wall of the intestine), with no evident tissue destruction, and were associated with the occurrence of hyperemia (i.e., engorgement of blood vessels with blood, giving the tissue a reddish coloration). The effects appeared at a spatial peak pressure amplitude of about 0.57 MPa for 1–2 min continuous exposures. Burst modes with 1-ms bursts repeated at 2– 4-ms intervals produced similar effects, which were comparable for comparable spatial average intensities. The petechiae observed to arise from heating with therapy-mode ultrasound were quite different from the intestinal hemorrhages induced by lithotripter shock waves. The shock-wave–induced hemorrhages involved blood flowing into the lumen of the intestine, with histologically obvious tissue destruction and clotting (Miller and Thomas 1995). The observation that ultrasound could produce vascular damage to the intestine both by heating (Miller and Thomas 1994) and by cavitation (Miller and Thomas 1995) led to an examination of the possibility of an interaction between the two mechanisms (Miller and Gies 1998a). Focused ultrasound at 400 kHz that was continuous or pulsed with 100 ms pulses, was applied to mice in a temperature-controlled water bath. Production of petechiae, which appeared to be primarily due to heating, and hemorrhages, which appeared to be primarily due to cavitation, were examined for various exposure conditions designed to elicit separate or combined operation of the two mechanisms. Petechia (up to about 100) occurred above 0.28 MPa (2.6 W cm22) for 1000 s continuous exposure at 37°C, and the threshold increased to 6.5 MPa (1.4 W cm temporal average) for 1000-s pulsed exposure (0.001 duty factor). Hemorrhages (up to about 10) were seen above 0.65 MPa for continuous exposure, and above 1.6 MPa for 1000-s pulsed exposure (0.001 duty factor). If a brief continuous exposure was broken into a low duty-factor pulsed exposure, the petechiae production decreased, but the number of hemorrhages increased. More petechia were induced at 42°C bath temperature relative to 32°C or 37°C, and the hemorrhage effect was somewhat enhanced by elevated tem-
Contrast agent enhances bioeffects ● D. L. MILLER and R. A. GIES
perature. Although some interaction could be discerned, for the most part, the two mechanisms appeared to act independently to produce vascular bioeffects on mouse intestine. The mouse intestine has become a valuable model system for ultrasound bioeffects research, and has been characterized in regard to vascular damage from both heating and cavitation under normal conditions. The intestines are especially convenient for studies of vascular damage because petechiae and hemorrhages can be counted by simple visual inspection without recourse to histology. The purpose of this present study was to utilize this model system to assess the possible association of vascular damage with ultrasonic activation of ultrasound contrast agent gas bodies circulating in the blood. With gas bodies present in the blood, the potential arises for damage, such as petechiae, to occur as a consequence of gas-body activation in addition to the thermal petechiae and nonthermal hemorrhage seen without added gas bodies. The addition of Albunex® to the circulation led to a complex interplay of thermal and nonthermal effects for different exposure conditions. METHODS Handling and treatment of the hairless male mice (Charles River, SK-H1) have been described previously (Miller and Thomas 1994), and all animal procedures were in accordance with institutional Animal Care Committee guidelines. A mouse was anesthetized with an intramuscular (IM) injection of 100 mg/kg ketamine (Ketaset®, Aveco Co., Fort Dodge, IA, USA) and 20 mg/kg xylazine (Rompun®, Mobay Corp., Shawnee, KA, USA). The animal was then mounted on a plastic board over a 2.5 cm diameter hole to allow passage of the beam, and placed at a 45° angle into a 37°C water bath. A rectal temperature probe (RET-3, Harvard Apparatus) was used to assure the equilibration of the mouse temperature with the bath temperature. Either Albunex® ultrasound contrast agent, or a gas-body–free blank, was introduced into the mouse by retro-orbital injection with a 25-G needle at a dosage of 10 mL/kg for most experiments immediately prior to exposure. Retro-orbital injection was chosen for introducing the agents into the blood, due to the rapid and positive injection afforded by this method (Bao et al. 1990). For this study, the Albunex® was purchased from Mallinckrodt Medical, St. Louis, MO, USA. The blank was a sterile 5% solution of human serum albumin (Sigma) in isotonic saline. When different dosages were used, a fraction of the blank agent was replaced with Albunex®, so that the injected volume was always the same. For exposure, an air-backed 2.5 cm diameter transducer was aimed upward at the beam hole at a 45° angle
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and positioned at 5.5 cm from the ventral surface of the mouse. The transducer was driven at 1.09 MHz, which was approximately its fundamental thickness resonance, by a signal generator (Model 3314A, Hewlett-Packard Co., Santa Clara, CA, USA) and amplifier (Model A-500, Electronic Navigation Industries, Rochester, NY, USA). A second oscillator (Model 33120A, Hewlett Packard) was used to gate the signal generator for some experiments. The exposure bath was filled with purified water, which was degassed for 1 h before each exposure session and filtered continuously through a 0.2 mm filter to minimize cavitation in the bath water. The ultrasound field was calibrated by measurements with a bilaminar shielded hydrophone with 0.5 mm sensitive spot (Marconi type Y-34-3598, National Physical Laboratory, Middlesex, UK). The 26 dB beam width was 13.4 mm at the mouse. Exposures were varied in 3-dB increments by linear extrapolation from the calibration at about 0.4 MPa. The influence of finite amplitude distortion on the beam was checked and the peak positive and peak negative differed by only about 20%, even for the highest exposures used in this study. The mean amplitude is specified to characterize the exposure level. After exposure, the mouse was removed from the bath and holder, and euthanized by CO2 asphyxiation at 5 min postexposure. The mouse was then dissected and the intestines were evaluated, as described previously (Miller and Gies 1998a). The number of petechiae, which were visible as small bright-red spots, extent of hyperemia, and the number of hemorrhages, which were visible as dark red blood in segments of the intestinal lumen, were evaluated and recorded. The petechiae, when present, had the same visual appearance regardless of the exposure mode, and no distinction could be made during scoring as to whether these lesions were caused by heating or by gas-body activation. Results are reported as the mean and standard error of 5 repeated measurements in different mice. Student’s t-tests were performed to compare results, with statistically significant difference assumed at p , 0.05 (two-sided test). Thresholds were estimated as the mean of the lowest pressure amplitude step with a statistically significant increase in a measure relative to 9 sham exposures, and the next lower step in the dose–response progression. Temperatures were measured in 4 separate mice (different from those used to study the bioeffects) using a 0.3-mm diameter thermocouple probe (model HYP-1, Omega Engineering) inserted into the abdomen immediately beneath the abdominal wall at the approximate position of the spatial peak intensity in the beam. These measurements should be considered estimates of the true maximum temperature elevations because the probe was not scanned to locate the exact maximum. Experiments were planned with continuous or pulsed-mode exposure
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with the goal of separating the effects of heating and gas-body activation. RESULTS Results for continuous 100-s exposures are shown in Fig. 1A, B, C for a range of pressure amplitudes. Hyperemia, an effect of heating, appears at about 0.5 MPa, and rises dramatically at 0.7 MPa, which was the maximum continuous-wave exposure level employed. Exposures with added contrast agent gas bodies enhanced this effect, for example, by a factor of 4 at 0.7 MPa. Petechiae were produced in the mouse intestine with the blank agent, and this effect was also enhanced by addition of gas bodies, for example, by a factor of about 5 at 0.7 MPa. No significant hemorrhage production occurred in the intestines for mice injected with the blank agent, but this effect was induced with Albunex® treatment. The thresholds determined for this exposureresponse experiment are listed in Table 1. For continuous exposure at 0.5 MPa, temperature elevations in 4 mice averaged 9.8°C (2.8°C SD). The thermal effects generally require some time to develop, but cavitational effects can presumably occur very quickly. The influence of the exposure duration on petechiae generation is shown in Fig. 2 for exposures with or without gas bodies. Hyperemia and hemorrhages were not appreciable except at 100 s (shown in Fig. 1). With Albunex®, significant petechia were produced at 10 s and 100 s. At 100 s, the petechiae production without added gas bodies was significantly different both from sham treatment and from the exposure with added gas bodies. Results for 100-s pulsed-mode exposures are shown in Fig. 1 for a range of pressure amplitudes of 10 ms pulses with a 1-ms pulse-repetition period (PRP). The pulse-mode exposure reduces the heating, and essentially no hyperemia was found for these exposures up to 2.8 MPa. Petechiae production was also minimal without added gas bodies, but hemorrhages began to appear at the highest exposure. Exposures with the contrast agent gas bodies began to yield significant numbers of petechiae at 1 MPa, but hemorrhage was seen only at the highest exposure. The petechiae seen with added gas bodies with pulsed exposure had the same visual appearance as the petechiae seen after the continuous exposures. The enhancement in the production of petechiae with added gas bodies was robust; for example, at 2.8 MPa, an average of 227 petechiae was produced with added gas bodies, a 30-fold increase. The thresholds determined for this exposure-response experiment are listed in Table 1. At 1.0 MPa with Albunex®, estimates of the temperature elevation from 4 mice averaged 0.6°C (0.3°C SD). The pronounced enhancement of petechiae produc-
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tion for pulse-mode exposure depends on the dosage of Albunex®, as shown in Fig. 3 for 2-MPa pulses. The line was fitted to the plotted data points by linear regression, with a slope of 10.4 petechia per mL/kg and an intercept of 6.4 petechia (r 2 5 0.97). Although there is a clear trend for increasing numbers of petechiae, statistically significant increases above the effect obtained with the blank agent were obtained only for the 5 mL/kg and 10 mL/kg doses. However, the petechia effect was significant relative to sham exposure for 0.1, 0.2, 1.0, 5.0 and 10 mL/kg contrast agent doses (but not for 0, 0.5 or 2.0 mL/kg). Hemorrhage and hyperemia were not appreciable in this series of exposures. The pulsing variables of pulse duration and duty factor were explored to provide a more complete picture of the role of pulsing in the effects with and without added contrast agent gas bodies. Results for 10-ms pulses (1 ms PRP) are compared with 1 ms pulses (100 ms PRP) in Fig. 4. Essentially, no effect was seen without Albunex® (10 mL/kg), and no difference was found in the petechiae counts for the two pulse durations. The range of pulse durations was extended, all with a 0.01 duty factor, further, to explore this variable. The results are shown in Fig. 5 for 10-ms to 100-ms pulses. All the gas-body agent results were significantly different from the blank agent results, except for the 10-ms pulse duration. The 100-ms duration pulses, which had a 10-s PRP giving only 10 pulses during the 100-s exposure, produced significantly more petechiae than the 1-ms or shorter pulses. The duty factor was also important because more closely spaced pulses increase the temporal average intensity (related to heating), as shown in Fig. 6 for 2-MPa, 10-ms pulses. The gas-body agent produced significantly more petechiae than the blank agent, but this enhancement declined greatly for the 0.001 duty cycle (10-ms PRP, 1000-s duration). The petechiae effect was also significantly different from sham exposure for the blank agent, at 0.1 duty factor. DISCUSSION The results of this study revealed a complex interplay of heating and gas-body activation in the etiology of ultrasound-induced intestinal effects. For continuous exposure, petechiae and hyperemia were induced even for the blank agent, and these effects were enhanced when gas bodies were injected (see Fig. 1). Because hyperemia was associated with the effects, heating must be a factor in production of the effects, as was noted previously for similar exposure conditions (Miller and Thomas 1994). The rapid decline in the effect for shorter exposure durations (see Fig. 2) also may be indicative of an important thermal role in the enhancement. The enhancement of the effects by the addition of contrast-agent gas
Contrast agent enhances bioeffects ● D. L. MILLER and R. A. GIES
Fig. 1. Results for average numbers (standard error bars) of (a) hyperemic length, (b) Petechiae and (c) Hemorrhages induced in intestines of mice exposed to continuous (circles) or pulsed (diamonds) ultrasound. Prior to exposure, mice received an injection of gas-body contrast agent (solid symbols) or blank agent without gas bodies (open symbols).
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Table 1. Thresholds, as defined in Methods, for petechiae and hemorrhages for each of the exposure conditions involving an exposure-response series without or with added gas bodies (1 GB). Threshold (MPa) Exposure condition Continuous Continuous 1 GB Pulsed 10 ms Pulsed 10 ms 1 GB Pulsed 1 ms Pulsed 1 ms 1 GB
Threshold (W cm22)
Petechia Hemorrhage Petechia Hemorrhage 0.4 0.4 1.2 0.8 1.7 0.4
ND 0.4 2.4 2.4 ND 1.7
5.3 5.3 0.48 0.21 0.96 0.05
ND 5.3 1.9 1.9 ND 0.96
The thresholds are specified in terms of the spatial peak pressure amplitude and the spatial peak; temporal average intensity calculated using the 0.01 duty factor for the pulsed modes. ND: not determinable from the data.
bodies may reflect an increase in absorption and heating, or the mechanical action of the gas bodies (or both). Theoretical considerations indicate that the presence of gas bodies or bubbles in tissue can increase the absorption and heating resulting from ultrasound exposure (Coakley and Nyborg 1978; Wu 1998). The significant production of hemorrhage into the intestinal lumen with added contrast agent suggests that the gas bodies may be initiating vigorous cavitation activity, because this effect was previously associated with lithotripter shock-wave exposure (Miller and Thomas 1995). For pulse-mode exposure, heating was minimized, hyperemia was avoided, and the petechia appeared with the addition of gas bodies at levels for which little or no effect was detectable for the blank agent. The cause of
Fig. 2. Results for average numbers (standard error bars) of petechiae for 0.7-MPa continuous exposures of varying duration with (solid circles) or without (open circles) added gas bodies.
Fig. 3. The average numbers (standard error bars) of petechiae obtained for various doses of contrast agent after exposure to 2-MPa pulsed ultrasound. The line was fitted to the data by linear regression (see text).
this effect was presumably the mechanical rupture of capillaries by the ultrasonically-activated gas bodies. The petechiae had the same visual appearance for continuous or pulsed exposures, and it was not possible to categorize these lesions as thermal or nonthermal in origin by simple visual inspection. The threshold for this pulsemode effect, 0.8 MPa peak pressure amplitude or 210 mW/cm2 SPTA intensity for the 10-ms pulses, lies within the range of parameters associated with diagnostic ultrasound.
Fig. 4. Results for average numbers (standard error bars) of petechiae produced by pulsed mode exposure for 10 ms or 1-ms pulses with 0.01 duty factor with or without added gas bodies.
Contrast agent enhances bioeffects ● D. L. MILLER and R. A. GIES
Fig. 5. Petechiae induced by 2-MPa pulsed ultrasound with various pulse durations, 0.01 duty cycle and 100-s duration, with and without added gas bodies.
In this study, the contrast agent was delivered as a per-kg dosage by retro-orbital injection. Unfortunately, the actual dosage of intact gas bodies reaching the general circulation and persisting until exposure (about 1 min) is unknown. This problem pertains to all small animal experimentation; for example, similar problems were encountered for tail-vein injection (Dalecki et al. 1997a). As a rough perspective, the dosage used for most experiments in this study was 10 mL/kg, which would extrapolate to a 1000-mL injection into a 100-kg human. The petechiae effect decreased roughly in proportion to the dosage (see Fig. 3). More realistic dosages, such as 0.1 mL/kg (10 mL in the 100-kg human) produced sta-
Fig. 6. Petechiae induced by 2-MPa pulsed ultrasound with 10 ms pulses and three different duty factors, with or without added contrast agent. The exposure duration was also varied to maintain a constant total on-time of 1 s.
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tistically significant, but greatly reduced, numbers of petechiae in this study. The results of pulsed-mode exposure in this study are different from those of the earlier study (Miller and Thomas 1994), which involved closely spaced 1-ms pulses. This difference was presumably due to the closely spaced pulses, which produced significant heating in the previous study, relative to the widely spaced pulses, which produced much less heating in this study. Pulsing parameters did not appear to be a strong factor in the exposure outcome; for example, results for 10-ms and 1-ms pulses were very similar (see Fig. 4). However, the threshold with gas bodies was lower for the longer pulse (50 mW/cm2, see Table 1). Very long, widely spaced pulses were more effective than short pulses at the same duty cycle (see Fig. 5). This phenomenon is puzzling because the longest pulses had a PRP of 10 s. This time was sufficient for the small gas bodies to dissolve and vanish after destabilization by the exposure and, thus, a diminution of the effect might have been anticipated for this condition. One explanation for this observation is that the long interpulse duration may have allowed inflow of a fresh supply of gas bodies from unexposed regions of the body. This concept has also been applied in diagnostic procedures, for which the ultrasound beam can reduce contrast (Porter et al. 1996). If unusually long times are allowed between scans, then the contrast effect returns. In conclusion, the addition of contrast agent during in vivo ultrasound exposure enhanced the production of vascular damage in intestinal tissue. In particular, added gas bodies yielded petechiae production under exposure conditions for which heating was insufficient to produced this effect. Thus, the petechia effect, as observed by visual inspection, can be produced by ultrasonic activation of blood-borne gas bodies, as well as by heating. The intestine was utilized for this study, because it is a previously examined model system, but other tissues would presumably also be effected. For example, petechiae were noted in the abdominal skin and muscle layers for many exposures yielding intestinal petechiae. The petechiae and hemorrhage effects were increased with added gas bodies for continuous exposure, which involved significant heating and hyperemia. For pulsed exposure, petechiae were produced for conditions yielding essentially no effect without the agent, presumably by the nonthermal mechanical action of the gas bodies. In this study, effects associated with the contrast agent were emphasized by utilizing relatively high doses of the agent, and effects decreased for lower dosages. Because the addition of gas bodies to the circulation introduces an extrinsic potential for activation of nonthermal mechanisms for bioeffects, some increased effectiveness might
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be expected for any dosage when the gas bodies interact with ultrasound. Acknowledgements—This investigation was supported by PHS Grant CA42947 awarded by the National Institutes of Health, DHHS.
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